Most flow cytometers are optimized for analysis of eukaryotic cells. Measurements of bacteria and viruses in conventional instruments are limited by photoelectron statistics; some microbial constituents, and smaller microbes, may thus be undetectable or not measurable with precision. In this project, a high-sensitivity, high-precision, slow flow multiparameter flow cytometer optimized for work with microorganisms will be built and tested. The apparatus will be sufficiently compact and user-friendly to be placed in environments in which it can be used in rapid identification and characterization of multiply resistant and bioengineered pathogens and in development of therapy for infections with these pathogens. The sensitivity of the apparatus for measurement of microbial nucleic acid content and cell-associated antigens or nucleic acid probes will be established, comparing single photon counting and conventional fluorescence detection methods. The precision of ratiometric measurements of bacterial pH, membrane potential and permeability, and, if possible, calcium ion content, will be evaluated and compared using slow-flow and conventional instruments, with a view toward refining cytometric assessment of drug effects on bacteria. The relationship between staining intensity and nucleic acid type and content in viruses, will be investigated, with a view toward refining cytometric virus detection and identification.